The regulation of glutamine synthesis in the food yeast Candida utilis: the purification and subunit structure of glutamine synthetase and aspects of enzyme deactivation.

Yeast glutamine synthetase was purified and shown to be an octameric globular protein (s = 15.4,fF0 = 1-29, mol. wt = 390000). It consists of two weakly-bound half molecules (s = 8.7, fFo = 1-35, mol. wt = 180500) and relatively harsh treatment is required to dissociate these octamers into component monomers (s = 3.8). Deactivation of the enzyme in vivo may include changes in the conformation of the native enzyme with its separation into two ' tight' tetramers, followed by their dis-sociation into component monomers and dimers. In the presence of Mg2+ and glutamate, monomers re-aggregate to oligomers which, although having transferase activity, are devoid of synthetase activity. The relevance of these observations in relation to control of glutamine synthetase activity in yeast is discussed. Previous studies on the glutamine synthetase of Candida utilis have revealed that the enzyme is subject to rapid and extensive deactivation (Ferguson & Sims, 1971). Modulation of enzyme concentration, achieved mainly by irreversible deactivation and rapid resynthesis, is primarily responsible for the regulation of glutamine synthesis (Sims & Ferguson, 1972; 1974; Ferguson & Sims, 1974a, b). These observations leave unanswered the question of how such precise adjustment of enzyme concentration is achieved. In the present paper we examine the subunit structure of the yeast glutamine synthetase and its possible influence on a mechanism for enzyme deactivation. Organism and culture conditions. Candida utilis NCYC 737 was grown as described earlier (Ferguson & Sims, 1971). Assay for enzyme activity. Glutamine synthetase [L-glutamate: ammonia ligase (ADP) EC. 6.3. I. 21 was measured as transferase activity and synthetase activity (see Ferguson & Sims, 1971; 1974~1). Measurement of radioactivity. The scintillation fluid contained 2,5-diphenyloxazole (3 g) and I ,~-bis-[2(~-methyl-~-phenyl-oxazol-2-yl)]benzene (0.3 g) in toluene (I 1) and Triton X-IOO (500 ml). Samples, made to I ml with water, were added to 12 ml of scintillation fluid and counted in a Packard Tricarb scintillation spectrometer. The d.p.m. were computed from the c.p.m. by a channel ratio method as described by Glass (1970). The efficiency of counting was about 70 %.